PROJECT SUMMARY The rising incidence of type 2 diabetes (T2D) highlights a growing need to understand mechanisms behind highly-effective therapies such as Roux-en-Y gastric bypass surgery (RYGB) in order to develop better, more widely-applicable treatments. The long-term goal is to understand the role of luminal factors to mediate the effect of intestinal re-routing upon energy metabolism, including T2D. The overall objective of this application is to test for tissue-level epigenetic changes in the intestine after RYGB, as a potential mechanism for Roux limb (RL) remodeling that we have shown to relate to T2D remission in humans. The central hypothesis is that RL metabolic reprogramming is driven by segment-specific epigenetic modifications which are plastic and require continuous luminal stimulation. The rationale for this project is that a greater understanding of intestinal gene expression regulatory networks after RYGB will shed light on ways to mimic RYGB?s effects with less invasive alternative therapies. The central hypothesis will be tested in two specific aims: (1) to test the role of an unexpected luminal nutrient load to influence RL epigenetic and transcriptomic signatures for key genes associated with metabolically-significant RL remodeling; and (2) to test the dependency of RL epigenetic signatures and metabolic substrate utilization upon in vivo factors using intestinal epithelial organoids (IEOs). In aim 1, gene expression (RNA-seq) and chromatin remodeling (ATAC-seq) will be studied in RL versus bypassed intestinal limb (biliopancreatic limb) or versus sleeve gastrectomy, and these methods will also be employed to test the plasticity of RL remodeling during caloric restriction and refeeding. In aim 2, IEOs will be used to ask whether epigenetic (ATAC-seq) and metabolic reprogramming (RNA-seq; Seahorse XF Analysis) change in the absence of in vivo factors. The candidate for this career development award, Dr. Margaret Stefater, is a pediatric endocrinologist with an MD/PhD and expertise in the integrated physiology of energy balance, especially after bariatric surgery. In order to achieve scientific independence for the candidate, a comprehensive career development plan has been developed to gain additional experience in the areas of (1) epigenetics and gene regulatory networks, (2) bioinformatics, and (3) use of intestinal organoids as a model of intestinal biology. This will be accomplished through coursework and seminars, under the mentorship of experts in the fields of bariatric surgery (Nicholas Stylopoulos) and intestinal biology (David Breault). The proposed research in this application is innovative, in the applicant?s opinion, because it represents a substantial departure from the status quo by using state-of-the-art sequencing and bioinformatics techniques to address how RYGB?s core surgical elements (e.g., biliary exclusion, accelerated nutrient delivery) influence epigenetic regulation of gene expression. Ultimately, this project will shed light on mechanisms linking luminal factors to known intestinal remodeling after RYGB, thus highlighting potential novel drug targets for the development of innovative T2D therapies, and paving the way for the applicant?s first R01 submission.